Drug-resistant Mycobacterium Tuberculosis Research Articles

Molecular epidemiology and drug resistance of Mycobacterium tuberculosis in a tertiary care hospital in northeastern Mexico.

Tuberculosis (TB) is a re-emerging disease considered a public health concern. In the present study, we analyzed the epidemiology and drug resistance of Mycobacterium tuberculosis strains isolated from patients with pulmonary TB. Mycobacterium tuberculosis isolates (n = 190) were obtained from patients with pulmonary TB admitted to Dr. José Eleuterio González University Hospital (UH). Each M. tuberculosis isolate was analyzed by spoligotyping (spacer oligonucleotide typing) and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat). Drug resistance was evaluated using the Anyplex™ II MTB/MDR/XDR assay. The predominant spoligotypes observed were X1 (SIT 119, n = 46), T1 (SIT 53, n = 40), H3 (SIT 50, n = 13), Beijing (SIT 1, n = 11), and EAI2-Manila (SIT 19, n = 8). MIRU-VNTR analysis showed that the locus QUB-26 had the highest allelic variability. The observed drug resistance included monoresistance to rifampicin (2.6%; n = 5), isoniazid (3.2%; n = 6), and fluoroquinolones (1.6%; n = 3) as well as multidrug resistance (5.3%; n = 10). All of the Beijing strains were susceptible. Regarding comorbidities, 13.7% (26/190) of the patients were co-infected with TB and HIV (TB+HIV+), and 31.6% (55/190) had TB along with diabetes (TB + diabetes). The most prevalent lineages were X1 (SIT 119; 24.3%) and T1 (SIT 53; 21%). An alarming proportion (12.6%) of M. tuberculosis isolates presented drug resistance. To effectively manage TB, continuous surveillance of regional strain dissemination, drug resistance profiles, and TB-associated comorbidities is crucial.

Изучение эффективности нового противотуберкулезного препарата тиозонид у пациентов с туберкулезом легких с множественной и широкой устойчивостью M. tuberculosis

Изучение эффективности нового противотуберкулезного препарата тиозонид у пациентов с туберкулезом легких с множественной и широкой устойчивостью M. tuberculosis

Virtual screening and identification of promising therapeutic compounds against drug-resistant Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase I (KasA)

The emergence of new Mycobacterium tuberculosis (Mtb) strains resistant to the key drugs currently used in the clinic for tuberculosis treatment can substantially reduce the probability of therapy success, causing the relevance and importance of studies on the development of novel potent antibacterial agents targeting different vulnerable spots of Mtb. In this study, 28,860 compounds from the library of bioactive molecules were screened to identify novel potential inhibitors of β-ketoacyl-acyl carrier protein synthase I (KasA), one of the key enzymes involved in the biosynthesis of mycolic acids of the Mtb cell wall. In doing so, we used a structure-based virtual screening approach to drug repurposing that included high-throughput docking of the C171Q KasA enzyme with compounds from the library of bioactive molecules including the FDA-approved drugs and investigational drug candidates, assessment of the binding affinity for the docked ligand/C171Q KasA complexes, and molecular dynamics simulations followed by binding free energy calculations. As a result, post-modeling analysis revealed 6 top-ranking compounds exhibiting a strong attachment to the malonyl binding site of the enzyme, as evidenced by the values of binding free energy which are significantly lower than those predicted for the KasA inhibitor TLM5 used in the calculations as a positive control. In light of the data obtained, the identified compounds are suggested to form a good basis for the development of new antitubercular molecules of clinical significance with activity against the KasA enzyme of Mtb. Communicated by Ramaswamy H. Sarma

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection.

This study aimed to evaluate the accuracy of detecting drug-resistant Mycobacterium tuberculosis complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS6110 and the mtp40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS6110 and mtp40 single-step PCR revealed the IS6110 in 27 of the 48 sputum samples, while the mtp40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.

Whole genome sequencing of drug-resistant Mycobacterium tuberculosis isolates in Victoria, Australia

ObjectivesWhole genome sequencing (WGS) can identify clusters, transmission patterns, and drug resistance mutations. This is important in low-burden settings such as Australia, as it can assist in efficient contact tracing and surveillance. MethodsWe conducted a retrospective cohort study using WGS from 155 genomically defined drug-resistant Mycobacterium tuberculosis (DR-TB) isolates collected between 2018-2021 in Victoria, Australia. Bioinformatic analysis was performed to identify resistance-conferring mutations, lineages, clusters and understand how local sequences compared with international context. ResultsOf the 155 sequences, 42% were identified as lineage 2 and 35% as lineage 1; 65.8% (102/155) were isoniazid mono-resistant, 8.4% were multi-drug resistant TB and 5.8% were pre-extensively drug-resistant / extensively drug-resistant TB. The most common mutations were observed in katG and fabG1 genes, especially at Ser315Thr and fabG1 -15 C>T for first-line drugs. Ser450Leu was the most frequent mutation in rpoB gene. Phylogenetic analysis confirmed that Victorian DR-TB were associated with importation events. There was little evidence of local transmission with only five isolate pairs. ConclusionIsoniazid-resistant TB is the commonest DR-TB in Victoria, and the mutation profile is similar to global circulating DR-TB. Most cases are diagnosed among migrants with limited transmission. This study highlights the value of WGS in identification of clusters and resistance-conferring mutations. This information is crucial in supporting disease mitigation and treatment strategies.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study

In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

A Single-Run HPLC-MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Asiaticoside A for the modulation of 1-TbAd- a potential target and ligand for extensive drug resistance Mycobacterium tuberculosis

In nature, terpene nucleosides are relatively rare, with 1-tuberculosinyladenosine (1-TbAd) being an exclusive feature of Mycobacterium tuberculosis (Mtb). The convergence of nucleosides and terpene pathways in the Mtb complex appears to have emerged late in its evolutionary history. 1-TbAd (PDB ID: 3WQK) is a prominent chemical marker for Mtb and may contribute to its virulence-related properties when exported extracellularly. We gathered a comprehensive set of 270 phytochemicals from diverse Ayurvedic texts and treatment traditions. Subsequently, we conducted structure-based molecular docking analyses to identify compounds exhibiting the strongest binding affinity for 1-TbAd, highlighting their potential as drug candidates. These selected compounds were further subjected to an in-vitro growth inhibition assay against the reference strain Mycobacterium tuberculosis h37rv. Among the candidates, Asiaticoside A (ASA) emerged as a promising candidate from the pool of 270 compounds. To assess the impact of ASA on 1-TbAd expression, we employed a PCR-based mRNA expression assay, revealing ASA's ability to downregulate 1-TbAd expression in extensively drug-resistant MTb strains. Remarkably, the conventional drug rifampin showed no such effectiveness in our experiments. We further conducted molecular dynamic simulations to explore the interaction between ASA and 1-TbAd in a cellular-like environment, confirming the stability of their interaction. Also, we predicted ASA's stability toward causing inducing the random mutations in the target gene. With this, we propose a novel target and its modulator to treat extensively drug-resistant MTB.Graphical

Genomic epidemiology analysis of drug-resistant Mycobacterium tuberculosis distributed in Mexico.

Genomics has significantly revolutionized pathogen surveillance, particularly in epidemiological studies, the detection of drug-resistant strains, and disease control. Despite its potential, the representation of Latin American countries in the genomic catalogues of Mycobacterium tuberculosis (Mtb), the bacteria responsible for Tuberculosis (TB), remains limited. In this study, we present a whole genome sequencing (WGS)-based analysis of 85 Mtb clinical strains from 17 Mexican states, providing insights into local adaptations and drug resistance signatures in the region. Our results reveal that the Euro-American lineage (L4) accounts for 94% of our dataset, showing 4.1.2.1 (Haarlem, n = 32), and 4.1.1.3 (X-type, n = 34) sublineages as the most prevalent. We report the presence of the 4.1.1.3 sublineage, which is endemic to Mexico, in six additional locations beyond previous reports. Phenotypic drug resistance tests showed that 34 out of 85 Mtb samples were resistant, exhibiting a variety of resistance profiles to the first-line antibiotics tested. We observed high levels of discrepancy between phenotype and genotype associated with drug resistance in our dataset, including pyrazinamide-monoresistant Mtb strains lacking canonical variants of drug resistance. Expanding the Latin American Mtb genome databases will enhance our understanding of TB epidemiology and potentially provide new avenues for controlling the disease in the region.

Effectiveness of Surgical Treatment of Patients with Respiratory Tuberculosis Complicated with Chronic Pleural Empyema Depending on Spread of Intrapleural Pathological Alterations

INTRODUCTION: Chronic tuberculous pleural empyema (CTPE) is a severe form of tuberculous lesion characterized by a purulent-destructive process in the residual pleural cavity, gross irreversible morphofunctional specific alterations in the pleura forming within three-five months after appearance of clinical signs of pleurisy. Patients with respiratory tuberculosis (RT) complicated with pleural empyema, are the most severe contingent of patients in tuberculosis (TB) hospitals.
 AIM: To study effectiveness of surgical treatment of patients with RT complicated with CTPE depending on spread of pathological alterations in the parietal pleura.
 MATERIALS AND METHODS: The study included 65 patients (43 men, 66.2%, aged 2361 years; 22 women, 33.8%, aged 2665 years) with RT complicated with CTPE, who underwent surgical treatment in the volume of pleurectomy with decortication of lung. Patients were divided to 3 groups depending on spread of pathological alterations in the parietal pleura: group 1 (n = 40, 61.5%) with spread of CTPE not exceeding the surface area of one anatomical wall of the pleural cavity; group 2 (n = 15, 23.1%) with CTPE involving 23 anatomical walls of the pleural cavity; group 3 (n = 10, 15.4%) with CTPE involving all anatomical walls of the pleural cavity.
 RESULTS: The effectiveness of complex treatment of patients with RT complicated with CTPE was 92.3% (n = 60). In the period from 2014 to 2021, the rate of postoperative complications after pleurectomy with decortication of lung including simultaneous resection, was 12.3%, mortality rate 0% and postoperative recurrences of empyema 7.7%. Despite adequate postoperative conservative treatment, the achievement of clinical effect and the absence of postoperative recurrences were directly proportional to the spread of intrapleural alterations. The destructive widespread forms of PT with multiple and wide-range drug resistance of mycobacterium tuberculosis are more common among the patients with subtotal and total forms of CTPE.
 CONCLUSION: The obtained results evidence a high effectiveness of surgical treatment of the given category of patients irrespective of spread of pathological alterations in the pleura and lung.

Clinical Whole-Genome Sequencing Assay for Rapid Mycobacterium tuberculosis Complex First-Line Drug Susceptibility Testing and Phylogenetic Relatedness Analysis.

The global rise of drug resistant tuberculosis has highlighted the need for improved diagnostic technologies that provide rapid and reliable drug resistance results. Here, we develop and validate a whole genome sequencing (WGS)-based test for identification of mycobacterium tuberculosis complex (MTB) drug resistance to rifampin, isoniazid, pyrazinamide, ethambutol, and streptomycin. Through comparative analysis of drug resistance results from WGS-based testing and phenotypic drug susceptibility testing (DST) of 38 clinical MTB isolates from patients receiving care in Los Angeles, CA, we found an overall concordance between methods of 97.4% with equivalent performance across culture media. Critically, prospective analysis of 11 isolates showed that WGS-based testing provides results an average of 36 days faster than phenotypic culture-based methods. We showcase the additional benefits of WGS data by investigating a suspected laboratory contamination event and using phylogenetic analysis to search for cryptic local transmission, finding no evidence of community spread amongst our patient population in the past six years. WGS-based testing for MTB drug resistance has the potential to greatly improve diagnosis of drug resistant MTB by accelerating turnaround time while maintaining accuracy and providing additional benefits for infection control, lab safety, and public health applications.

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Genomic Characterization of Drug-Resistant Mycobacterium tuberculosis L2/Beijing Isolates from Astana, Kazakhstan.

Kazakhstan ranks among the countries with the highest number of MDR-TB patients per 100,000 population worldwide. The successful transmission of local MDR strains of Mycobacterium tuberculosis (Mtb) poses a significant threat to disease control. In this study, we employed whole-genome sequencing to examine drug resistance, compensatory mutations, population structure, and transmission patterns in a sample of 24 clinical isolates of L2/Beijing Mtb collected in Astana, Kazakhstan between 2021 and 2022. The genotypic prediction of Mtb susceptibility to anti-TB agents was consistent with the phenotypic susceptibility, except for bedaquiline. An analysis of resistance-associated genes characterized most of the isolates as pre-extensively drug-resistant tuberculosis (pre-XDR-TB) (n = 15; 62.5%). The phylogenetic analysis grouped the isolates into four transmission clusters; the dominant cluster was assigned to the "aggressive" Central Asia outbreak (CAO) clade of L2/Beijing (n = 15; 62.5%). Thirteen mutations with putative compensatory effects were observed exclusively in Mtb isolates containing the rpoB S450L mutation. The putative compensatory mutations had a stabilizing effect on RpoABC protein stability and dynamics. The high prevalence of the CAO clade in the population structure of Mtb may explain the rapid spread of MDR-TB in Kazakhstan.

Mixed infections in genotypic drug-resistant Mycobacterium tuberculosis

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major global public health problem, resulting in more than 1 million deaths each year. Drug resistance (DR), including multi-drug (MDR-TB), is making TB control difficult and accounts for 16% of new and 48% of previously treated cases. To further complicate treatment decision-making, many clinical studies have reported patients harbouring multiple distinct strains of M. tuberculosis across the main lineages (L1 to L4). The extent to which drug-resistant strains can be deconvoluted within mixed strain infection samples is understudied. Here, we analysed M. tuberculosis isolates with whole genome sequencing data (n = 50,723), which covered the main lineages (L1 9.1%, L2 27.6%, L3 11.8%, L4 48.3%), with genotypic resistance to isoniazid (HR-TB; n = 9546 (29.2%)), rifampicin (RR-TB; n = 7974 (24.4%)), and at least MDR-TB (n = 5385 (16.5%)). TB-Profiler software revealed 531 (1.0%) isolates with potential mixed sub-lineage infections, including some with DR mutations (RR-TB 21/531; HR-TB 59/531; at least MDR-TB 173/531). To assist with the deconvolution of such mixtures, we adopted and evaluated a statistical Gaussian Mixture model (GMM) approach. By simulating 240 artificial mixtures of different ratios from empirical data across L1 to L4, a GMM approach was able to accurately estimate the DR profile of each lineage, with a low error rate for the estimated mixing proportions (mean squared error 0.012) and high accuracy for the DR predictions (93.5%). Application of the GMM model to the clinical mixtures (n = 531), found that 33.3% (188/531) of samples consisted of DR and sensitive lineages, 20.2% (114/531) consisted of lineages with only DR mutations, and 40.6% (229/531) consisted of lineages with genotypic pan-susceptibility. Overall, our work demonstrates the utility of combined whole genome sequencing data and GMM statistical analysis approaches for providing insights into mono and mixed M. tuberculosis infections, thereby potentially assisting diagnosis, treatment decision-making, drug resistance and transmission mapping for infection control.

Mycobacteriophage D29 Lysin B exhibits promising anti-mycobacterial activity against drug-resistant Mycobacterium tuberculosis.

The emergence of antibiotic resistance in Mycobacterium tuberculosis has been the culprit behind the inefficient treatment of tuberculosis (TB). Mycobacteriophages being rapid and specific in their activity against mycobacteria including M. tuberculosis are an emerging alternative solution to the antibiotic resistance problem. D29 mycobacteriophage is known as an efficient lytic phage. Its lytic cassette encodes for two lytic proteins, Lysin A (LysA) and Lysin B (LysB), and a holin protein responsible for the translocation of the former two. LysB is a mycolyl-arabinogalactan esterase, which has shown a promising lytic effect on M. tuberculosis. In this study, we explored the lytic efficiency of D29 mycobacteriophage LysB protein on drug-resistant strains of M. tuberculosis. We demonstrate that LysB effectively lyses multi-drug-resistant M. tuberculosis both alone and along with anti-TB drugs. We also report that the surfactant Tween-80, although not vital, potentiates LysB activity. We are able to demonstrate that LysB can eliminate M. tuberculosis residing in mouse macrophages. Thus, by combining our in vitro and ex vivo data, we are able to claim that LysB is a potential phage-derived therapeutic which should undergo pre-clinical and clinical trials. IMPORTANCE To combat the rapidly emerging drug-resistant M. tuberculosis, it is now essential to look for alternative therapeutics. Mycobacteriophages can be considered as efficient therapeutics due to their natural ability to infect and kill mycobacteria including M. tuberculosis. Here, we have exploited the mycolyl-arabinogalactan esterase property of LysB encoded from mycobacteriophage D29. This study is novel in terms of targeting a multi-drug-resistant pathogenic strain of M. tuberculosis with LysB and also examining the combination of anti-TB drugs and LysB. All the experiments include external administration of LysB. Therefore, the remarkable lytic activity of LysB overcomes the difficulty to enter the complex cell envelope of mycobacteria. Targeting the intracellularly located M. tuberculosis by LysB and non-toxicity to macrophages take the process of the development of LysB as a drug one step ahead, and also, the interaction studies with rifampicin and isoniazid will help to form a new treatment regimen against tuberculosis.

Distribution of multi-drug resistant tuberculosis in Ekiti and Ondo states, Nigeria

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the top infectious killer diseases in the world. The emergence of drug-resistant MTB strains has thrown challenges in controlling TB worldwide. This study investigated the prevalence of drug-resistant tuberculosis in the states of Nigeria and the risk factors that can increase the incidence of tuberculosis. MethodsThe study is a cross-sectional epidemiological research carried out in the six senatorial districts of Ekiti and Ondo states, Nigeria, between February 2019 and January 2020. A structured questionnaire was administered to 1203 respondents for socio-demographic information, and sputum samples were collected from them for TB investigation. GeneXpert technique was used to diagnose TB from the sputum samples, followed by bacterial isolation using Löweinstein-Jensen medium and antibiotic susceptibility testing. ResultsPrevalence of TB in the two states combined was 15 ​%; with 13.8 ​% for Ekiti state and 16.1 ​% for Ondo State. The distribution of TB in the senatorial districts was such that: Ondo South ​> ​Ekiti Central ​> ​Ekiti South ​> ​Ondo North ​> ​Ekiti North ​> ​Ondo Central. The risk factors identified for TB prevalence in two states were gender, male ​> ​female (OR ​= ​0.548, p ​= ​0.004); overcrowding (OR ​= ​0.733, p ​= ​0.026); room size (OR ​= ​0.580, p ​= ​0.002); smoking (OR ​= ​0.682, p ​= ​0.019) and dry and dusty season (OR ​= ​0.468, p ​= ​0.005). The prevalence of MDR-TB in Ekiti and Ondo States were 1.2 ​% and 1.3 ​% respectively. The identified risk factors for MDR were education (OR ​= ​0.739, p ​= ​0.017), age (OR ​= ​0.846, p ​= ​0.048), religion (OR ​= ​1.95, p ​= ​0.0003), family income (OR ​= ​1.76, p ​= ​0.008), previous TB treatment (OR ​= ​3.64, p ​= ​0.004), smoking (OR ​= ​1.33, p ​= ​0.035) and HIV status (OR ​= ​1.85, p ​= ​0.006). Rifampicin monoresistant was reported in 6.7 ​% of the rifampicin-resistant strains, while 93.3 ​% were rifampicin polyresistant strains. Two (13.3 ​%) of the MDR-TB strains were resistant to all the 3 first-line antimycobacterial agents. All the Rifampicin-resistant TB strains were susceptible to the aminoglycosides (Amikacin, Capreomycin and Kanamycin), also with high susceptibility to the fluoroquinilones: Moxifloxacin (100 ​%) and Levofloxacin (86.7 ​%). Sixteen (94.1 ​%) of the 17 Rifampicin-susceptible strains were susceptible to all the eight antibiotics tested, while one (5.9 ​%) was susceptible to Rifampicin and Isoniazid but resistant to the rest antibiotics. Conclusion: The study showed that there is high prevalence of TB and MDR-TB in Ekiti and Ondo States Nigeria, hence, to meet the SDG Target 3.3 of ending TB epidemic by 2030, culturing and antibiotic susceptibility testing should be carried out on every TB-positive sputum and the patients treated accordingly.

Mycobacterium tuberculosis Sub-Lineage 4.2.2/SIT149 as Dominant Drug-Resistant Clade in Northwest Ethiopia 2020-2022: In-silico Whole-Genome Sequence Analysis.

Drug resistance (DR) in Mycobacterium tuberculosis complex (MTBC) is mainly associated with certain lineages and varies across regions and countries. The Beijing genotype is the leading resistant lineage in Asia and western countries. M. tuberculosis (Mtb) (sub) lineages responsible for most drug resistance in Ethiopia are not well described. Hence, this study aimed to identify the leading drug resistance sub-lineages and characterize first-line anti-tuberculosis drug resistance-associated single nucleotide polymorphisms (SNPs). A facility-based cross-sectional study was conducted in 2020-2022 among new and presumptive multidrug resistant-TB (MDR-TB) cases in Northwest Ethiopia. Whole-genome sequencing (WGS) was performed on 161 isolates using Illumina NovaSeq 6000 technology. The SNP mutations associated with drug resistance were identified using MtbSeq and TB profiler Bioinformatics softwares. Of the 146 Mtb isolates that were successfully genotyped, 20 (13.7%) harbored one or more resistance-associated SNPs. L4.2.2.ETH was the leading drug-resistant sub-lineage, accounting for 10/20 (50%) of the resistant Mtb. MDR-TB isolates showed extensive mutations against first-line anti-TB drugs. Ser450Leu/(tcg/tTg) for Rifampicin (RIF), Ser315Thr/(agc/aCc) for Isoniazid (INH), Met306Ile/(atg/atA(C)) for Ethambutol (EMB), and Gly69Asp for Streptomycin (STR) were the leading resistance associated mutations which accounted for 56.5%, 89.5%, 47%, and 29.4%, respectively. The presence of both clustered and non-clustered drug resistance (DR) isolates indicated that the epidemics is driven by both new DR development and acquired resistance. The high prevalence of drug-resistant TB due to geographically restricted sub-lineages (L4.2.2.ETH) indicates the ongoing local micro epidemics. The Mtb drug resistance surveillance system must be improved. Further evolutionary analysis of L4.2.2.ETH strain is highly desirable to understand evolutionary forces that leads L4.2.2.ETH in to high level DR and transmissible sub-lineage.

Multi-epitope vaccine against drug-resistant strains of Mycobacterium tuberculosis: a proteome-wide subtraction and immunoinformatics approach.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Toll-like receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

In vitro activities of contezolid (MRX-I) against drug-sensitive and drug-resistant Mycobacterium tuberculosis.

A novel oxazolidinone for the treatment of Mycobacterium tuberculosis has been developed, but the activity of contezolid (MRX-I) still needs to be clarified. In this study, we isolated Mycobacterium tuberculosis from 48 clinical patients with pulmonary tuberculosis. Roche drug susceptibility tests identified drug-sensitive and 39 drug-resistant M. tuberculosis isolates. Drug susceptibility assays indicated that MRX-I exhibited anti-Mycobacterium tuberculosis activity against both drug-sensitive and drug-resistant isolates, with an advantage against drug-resistant isolates. The results also showed that the anti-Mycobacterium tuberculosis activity was comparable to that of linezolid. IMPORTANCE Currently, Mycobacterium tuberculosis has exhibited increased drug resistance, leading to ineffective drug treatment in many patients with tuberculosis. Among the anti-Mycobacterium tuberculosis drugs, oxazolidinones have been gradually developed. Contezolid (MRX-I) has been newly developed in China with advantages versus the first oxazolidinone antibiotic approved by the Food and Drug Administration for clinical use, but the anti-M. tuberculosis activity needs to be further clarified. In this study, in vitro activities of MRX-I against M. tuberculosis were tested. The drug susceptibility assays indicated that MRX-I exhibited anti-M. tuberculosis activity comparable to that of linezolid, with an advantage against drug-resistant isolates.